JP2001258549A - Lactobacillus acidophilus hy2177 and lactobacillus casei hy2743 having antibacterial activity against helicobacter pylori, lactic acid bacterium preparation and fermented milk using the same - Google Patents
Lactobacillus acidophilus hy2177 and lactobacillus casei hy2743 having antibacterial activity against helicobacter pylori, lactic acid bacterium preparation and fermented milk using the sameInfo
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- lactobacillus acidophilus
- scabii
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/205—Bacterial isolates
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/23—Lactobacillus acidophilus
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は胃潰瘍の原因菌とし
て知られた抗菌活性を有するラクトバシルスアシドフィ
ルス(アシドフィルス菌)HY 2177、ラクトバシ
ルスカセイ(カセイ菌)HY 2743及びそれを用い
た乳酸菌製剤と発酵乳に関する。TECHNICAL FIELD The present invention relates to Lactobacillus acidophilus (Acidophilus) HY 2177, Lactobacillus sacchari (Casaceae) HY 2743 having antibacterial activity known as causative bacteria of gastric ulcer, and lactic acid bacteria preparations using the same. And fermented milk.
【0002】[0002]
【従来の技術】一般に、慢性胃炎、十二指腸疾病と密接
に関連があるものとして知られたヘリコバクターピロリ
は、1983年ウォレンとマーシャルが患者の胃幽門か
ら分離して報告した(J.R.Warren and
B.J.Marshall:Lancet、1273〜
1275、1988)。前記ヘリコバクターピロリは胃
粘膜上皮細胞間接合部で棲息しながら慢性胃潰瘍を誘発
するグラム陰性肝菌で、胃粘膜で湾曲型又はS字形の肝
菌として観察され、その大きさは0.4〜1.25μm
程度である。ヘリコバクターピロリの最も特異な生化学
的特性は尿素をアンモニアと二酸化炭素に分解する酵
素、ウレアアミドヒドロラーゼであるウレアーゼの非常
に強い生産能があることである。したがって、前記ヘリ
コバクターピロリは、胃粘膜の血漿滲出液又は組織液内
の尿素を分解して菌体周囲をアルカリ性にすることで、
胃内の強酸を中和させるものと知られている。BACKGROUND OF THE INVENTION Helicobacter pylori, generally known to be closely associated with chronic gastritis and duodenal disease, was reported by Warren and Marshall in 1983, isolated from the gastric pylorus of a patient (JR Warren and
B. J. Marshall: Lancet, 1273-
1275, 1988). Helicobacter pylori is a gram-negative hepatic bacterium that induces chronic stomach ulcer while living at the junction between gastric mucosal epithelial cells, and is observed as a curved or S-shaped hepatic bacterium in the stomach mucosa and has a size of 0.4-1. .25 μm
It is about. The most specific biochemical property of Helicobacter pylori is its ability to produce urease, a urea amide hydrolase, an enzyme that breaks down urea into ammonia and carbon dioxide. Therefore, the Helicobacter pylori decomposes urea in the plasma exudate or tissue fluid of the gastric mucosa to make the surrounding cells alkaline,
It is known to neutralize strong acids in the stomach.
【0003】前記ヘリコバクターピロリの他の特徴は、
菌体の一側面又は両側面に膜で取り囲まれた鞭毛(fl
agella)が一つずつ又は糸塊として存在している
ため、ねっとりした環境でも移動できることである。ヘ
リコバクターピロリの運動性は大腸菌が動けない粘度を
有する粘液より約20倍以上の粘液でも動けるものと知
られている。このような特性のため、サリチル酸塩(s
alicylate)又はエタノールのような化合物以
外では浸透しにくい胃粘液層を貫いて入ることができ、
粘液内でも自由に移動しながら胃上皮細胞の接合部に容
易に付着できるものと知られている。[0003] Other features of the Helicobacter pylori are:
Flagella (fl) surrounded by a membrane on one or both sides of the cells
aggella) can be moved in a soggy environment because they are present one by one or as a lump. It is known that the motility of Helicobacter pylori can move even with mucus about 20 times or more higher than mucus having a viscosity that Escherichia coli cannot move. Because of these properties, salicylates (s
alicylate) or a compound such as ethanol, which can penetrate the gastric mucus layer which is difficult to penetrate,
It is known that it can easily adhere to the junction of gastric epithelial cells while freely moving in mucus.
【0004】ヘリコバクターピロリの正確な汚染経路又
は伝染源については未だに正確に証明されてはいない
が、経口的方法により伝達、感染されるものと推定され
ている。また、成人のおよそ80%程度がこの菌に感染
されているが、臨床症状を示していないし、いずれかの
発病促進因子の影響により慢性的な胃潰瘍、十二指腸潰
瘍を誘発するものと知られている。この胃潰瘍、胃炎、
十二指腸潰瘍の治療薬としては、シメチジン、ラニチジ
ン、ファモチジンなどのH2受容体拮抗薬、オメプラゾ
ールなどのプロトンポンプ阻害薬などがある。これらは
治療効果は高いが、主として胃酸分泌抑制に対する薬理
作用を持っているため、再発率も高い。これは、ヘリコ
バクターピロリを直接除去していないためであると推定
されている。[0004] Although the exact route of contamination or the source of transmission of Helicobacter pylori has not yet been proven precisely, it is presumed that it will be transmitted and transmitted by oral means. Approximately 80% of adults are infected with this bacterium, but do not show clinical symptoms, and are known to induce chronic gastric ulcer and duodenal ulcer due to the effects of any oncolytic factor. . This gastric ulcer, gastritis,
The therapeutic agent for duodenal ulcer, cimetidine, ranitidine, H 2 receptor antagonists such as famotidine, and the like proton pump inhibitors such as omeprazole. Although they have a high therapeutic effect, they have a high recurrence rate mainly because they have a pharmacological effect on gastric acid secretion suppression. It is presumed that this is because Helicobacter pylori was not directly removed.
【0005】次いで、1994年2月米国国立衛生研究
所(NIH)では、ヘリコバクターピロリ感染患者に初
期又は再発の有無にかかわらず、胃酸分泌抑制剤に加え
て抗菌剤治療を勧告している。また、ヘリコバクターピ
ロリの除菌が消化性潰瘍の新たな治療法として台頭して
いる。更に、最近にはビスマス製剤、アモキシシリン、
メトロニダゾール又はビスマス製剤、テトラサイクリ
ン、メトロニダゾールを消化性潰瘍患者に同時に投与す
る三重療法により80%内外の治療効果を得たという報
告がある。[0005] Next, in February 1994, the National Institutes of Health (NIH) recommended treatment of Helicobacter pylori-infected patients with antibacterial agents in addition to gastric acid secretion inhibitors, regardless of whether they were initial or had recurrence. Helicobacter pylori eradication is emerging as a new treatment for peptic ulcers. More recently, bismuth preparations, amoxicillin,
There is a report that a triple therapy in which a metronidazole or bismuth preparation, tetracycline, or metronidazole is simultaneously administered to peptic ulcer patients has achieved a therapeutic effect of about 80% inside and outside.
【0006】しかし、前記抗生物質治療は、一般の感染
症の治療に使用される抗生物質より多くの量が処方さ
れ、投与期間もたいてい長期間であるため、新たな耐性
菌株が現れる危険性が高く、副作用の発生可能性も内在
する。[0006] However, since the antibiotic treatment is prescribed in a larger amount than the antibiotics used for the treatment of general infectious diseases and the administration period is usually long, there is a risk that a new resistant strain will appear. High, with the potential for side effects.
【0007】したがって、最近では、抗生物質に代わっ
て天然素材又は乳酸菌を用いるヘリコバクターピロリの
除菌が試験されている。タバク(Tabak)などはタ
イム(Thyme)から(J.of Applied
Bacteriogy,1996,80,667〜67
2)、ディカー(Diker)とハセリック(Hasc
elik)は茶からヘリコバクターピロリに対する抗菌
活性を報告し(Letters in Applied
Microbiology,1994,19,299
〜300)、ミドロ(Midolo)などは乳酸菌から
(J. ofApplied Bacteriolog
y,1995,79,475〜479)、バチア(Bh
atia)などはラクトバシルスアシドフィルス(L.
acidophilus)からヘリコバクターピロリに
対する抗菌活性を確認した(J.of Clinica
l Microbiology,1989,Oct.,
2328〜2330)。ネスラーはラクトバシルスアシ
ドフィルス菌株をヘリコバクターピロリ除菌のための抗
胃炎薬、抗潰瘍薬に用いたが(JP 94−9878
2)、バゾリ(Bazzoli)などは20人を対象と
した臨床実験でネスラーのラクトバシルスアシドフィル
スを8週間にわたり長期投与した結果、ヘリコバクター
ピロリの除菌に有効な結果が得られなかったと報告した
(Gastroenterology,1992,10
2,A38)。ワカモト製薬株式会社はプロバイオティ
ックスとしてラクトバシルスサリバリウスとラクトバシ
ルスブレビス(乳酸短杆菌)を抗胃炎薬、抗潰瘍薬及び
発酵食品の有効成分に用いた(JP97−24117
3)。[0007] Therefore, recently, eradication of Helicobacter pylori using natural materials or lactic acid bacteria instead of antibiotics has been tested. Tabak etc. are based on Time (Thyme) (J. of Applied)
Bacteriology, 1996, 80, 667-67.
2), Diker and Haseric (Hasc)
elik) reported antibacterial activity against Helicobacter pylori from tea (Letters in Applied).
Microbiology, 1994, 19, 299
~ 300), Midoro and the like from lactic acid bacteria (J. of Applied Bacteriology).
y, 1995, 79, 475-479), Bachia (Bh
atia) and the like are used in Lactobacillus acidophilus (L.
acidophilus), the antibacterial activity against Helicobacter pylori was confirmed (J. of Clinica).
l Microbiology, 1989, Oct. ,
2328-2330). Nestler used Lactobacillus acidophilus strain as an anti-gastritis drug and an anti-ulcer drug for eradication of Helicobacter pylori (JP 94-9878).
2), Bazzoli et al. Reported in a clinical experiment on 20 people that long-term administration of Nesler's Lactobacillus acidophilus for 8 weeks resulted in no effective results for eradication of Helicobacter pylori ( Gastroenterology, 1992, 10
2, A38). Wakamoto Pharmaceutical Co., Ltd. used Lactobacillus salivarius and Lactobacillus brevis (short lactobacilli) as probiotics as anti-gastritis drugs, anti-ulcer drugs and active ingredients in fermented foods (JP97-24117).
3).
【0008】しかし、前記特許の場合は、胃内定着性と
胃炎発生メカニズムのインターロイキン−8生成抑制に
よる結果で、ヘリコバクターピロリに対する抗菌活性又
はウレアーゼ活性抑制などヘリコバクターピロリに対す
る直接的な除菌効果にはアプローチできない問題点があ
った。[0008] However, in the case of the above-mentioned patent, the results of the inhibition of interleukin-8 production in the stomach and the mechanism of gastritis generation have a direct effect on the antibacterial activity against Helicobacter pylori or the direct eradication effect on Helicobacter pylori such as the inhibition of urease activity. There was a problem that could not be approached.
【0009】[0009]
【発明が解決しようとする課題】本発明は前記問題点に
鑑みてなされたもので、本発明の目的は、胃炎、胃潰瘍
の原因菌として知られたヘリコバクターピロリに対する
抗菌活性、ウレアーゼ活性抑制、胃内定着抑制、インタ
ーロイキン−8生成抑制の能力を有する乳酸菌を提供
し、これを有効成分とする胃炎、胃潰瘍、十二指腸潰瘍
の予防薬と治療薬を提供することにある。DISCLOSURE OF THE INVENTION The present invention has been made in view of the above problems, and an object of the present invention is to provide an antibacterial activity against Helicobacter pylori, which is known to cause gastritis and gastric ulcer, an inhibition of urease activity, An object of the present invention is to provide a lactic acid bacterium having the ability to inhibit internal colonization and inhibit the production of interleukin-8, and to provide a preventive and therapeutic agent for gastritis, gastric ulcer, and duodenal ulcer using the same as an active ingredient.
【0010】[0010]
【課題を解決するための手段】前記目的を達成するた
め、本発明のヘリコバクターピロリに対して抗菌活性を
有するラクトバシルスアシドフィルスHY2177、ラ
クトバシルスカセイHY2743及びそれを用いる製剤
は、人間から分離、保存されているラクトバシルス属、
ビフィドバクテリウム属、エンテロコッカス属の乳酸菌
のうち、韓国の胃潰瘍患者から分離したヘリコバクター
ピロリKS 51菌株に対する抗菌活性を有する乳酸菌
を選択し、前記選択された乳酸菌に対してヘリコバクタ
ーピロリのウレアーゼ活性抑制能力と胃内定着性抑制能
力を有する乳酸菌を再選択し、ヘリコバクターピロリに
よる胃炎発生メカニズムに関係するインターロイキン−
8の生成抑制能力を比較測定して最終的に選択したもの
である。In order to achieve the above object, Lactobacillus acidophilus HY2177 and Lactobacillus scabii HY2743 having antibacterial activity against Helicobacter pylori of the present invention, and a preparation using the same are separated from humans. Lactobacillus sp.
Among the lactic acid bacteria belonging to the genus Bifidobacterium and Enterococcus, lactic acid bacteria having an antibacterial activity against Helicobacter pylori KS51 strain isolated from a patient with gastric ulcer in Korea are selected, and the ability of Helicobacter pylori to inhibit the urease activity of the selected lactic acid bacteria is selected. And a lactic acid bacterium having the ability to inhibit gastric colonization, and interleukin involved in the mechanism of gastritis caused by Helicobacter pylori-
8 was finally selected by comparing and measuring the production inhibiting ability.
【0011】[0011]
【発明の実施の形態】以下、添付図面に基づいて本発明
をより詳細に説明する。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.
【0012】本発明者らは人間から分離、保存していた
ラクトバシルス属、ビフィドバクテリウム属、エンテロ
コッカス属の乳酸菌のうち、韓国の胃潰瘍患者から分離
したヘリコバクターピロリKS 51菌株に対する抗菌
活性を有する乳酸菌を選択した。この乳酸菌に対してヘ
リコバクターピロリのウレアーゼ活性抑制能力と胃内定
着性抑制能力を有する乳酸菌を再選択した。そして、ヘ
リコバクターピロリによる胃炎発生メカニズムに関係す
るインターロイキン−8の生成抑制能力を比較測定し、
ラクトバシルスアシドフィルスHY 2177(Lac
tobacillus acidophilus HY
2177, 寄託機関:韓国種菌協会,寄託番号:K
FCC 11142号, 寄託年月日:2000.2.
11.)とラクトバシルスカセイHY 2743(La
ctobacillus casei HY 274
3,寄託機関:韓国種菌協会,寄託番号:KFCC 1
1143,寄託年月日:2000.2.11.)を最終
的に選択した。Among the lactic acid bacteria of the genus Lactobacillus, Bifidobacterium and Enterococcus isolated and preserved from humans, the present inventors have found that lactic acid bacteria having antibacterial activity against Helicobacter pylori KS51 strain isolated from patients with gastric ulcer in Korea Was selected. Lactic acid bacteria having the ability to inhibit the urease activity of Helicobacter pylori and the ability to inhibit gastric colonization were selected again for the lactic acid bacteria. Then, the ability to inhibit the production of interleukin-8 related to the mechanism of gastritis caused by Helicobacter pylori was measured comparatively,
Lactobacillus acidophilus HY 2177 (Lac
tobacillus acidophilus HY
2177, Depositary institution: Korean Species Association, Deposit No .: K
FCC 11142, Deposit date: 2000.2.
11. ) And Lactobacillus kassay HY 2743 (La
ctobacillus casei HY 274
3. Depositary institution: Korean Seed Association, Deposit No .: KFCC 1
1143, Date of deposit: 2000.2.11. ) Was finally selected.
【0013】菌株特性 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743をMRS培地に37℃で2
4時間培養したときの生理学的特性を下記の表1に示し
た。Strain characteristics Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY2743 were added to MRS medium at 37 ° C. for 2 hours.
The physiological characteristics when cultured for 4 hours are shown in Table 1 below.
【0014】[0014]
【表1】 [Table 1]
【0015】抗菌活性 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743を12%脱脂粉乳に0.1
%接種し37℃で24時間培養した後、抑制環方法によ
りヘリコバクターピロリに対する抗菌活性を測定した。
その結果、ラクトバシルスアシドフィルスHY 217
7はおよそ18.9±1.5mmの抑制環、ラクトバシ
ルスカセイHY 2743はおよそ14.2±1.0m
mの抑制環を現することから、ほかの乳酸菌に比べヘリ
コバクターピロリに対する抗菌活性が高い(後述する実
施例1参照)。Antibacterial activity Lactobacillus acidophilus HY2177 and Lactobacillus scabii HY2743 are added to 12% skim milk powder in an amount of 0.1%.
% Inoculated and cultured at 37 ° C. for 24 hours, and then the antibacterial activity against Helicobacter pylori was measured by the inhibition ring method.
As a result, Lactobacillus acidophilus HY217
7 is a restraining ring of about 18.9 ± 1.5 mm, Lactobacillus kassay HY 2743 is about 14.2 ± 1.0 m
Since it exhibits an inhibitory ring of m, it has a higher antibacterial activity against Helicobacter pylori than other lactic acid bacteria (see Example 1 described later).
【0016】また、ラクトバシルスアシドフィルスHY
2177とラクトバシルスカセイHY 2743の混
合培養はヘリコバクターピロリに対する抗菌活性が単独
培養に比べて抗菌活性があるため、相乗効果をもたらす
ことを確認した(後述する実施例6参照)。Also, Lactobacillus acidophilus HY
It was confirmed that the mixed culture of 2177 and Lactobacillus scabii HY 2743 has a synergistic effect since the antibacterial activity against Helicobacter pylori is higher than that of the single culture (see Example 6 described later).
【0017】ヘリコバクターピロリウレアーゼ活性抑制
効果 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743を12%脱脂粉乳に0.1
%接種し37℃で24時間培養した後、ΔpH方法によ
りヘリコバクターピロリウレアーゼ活性抑制効果を測定
した。ラクトバシルスアシドフィルスHY 2177と
ラクトバシルスカセイHY 2743がヘリコバクター
ピロリウレアーゼ活性をおよそ90%程度抑制すること
が示された(後述する実施例2参照)。Helicobacter pylori urease activity inhibitory effect Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were added to 12% skim milk powder at a concentration of 0.1%.
% Inoculated and cultured at 37 ° C. for 24 hours, and then the effect of inhibiting Helicobacter pylori urease activity was measured by the ΔpH method. It was shown that Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 suppress Helicobacter pylori urease activity by about 90% (see Example 2 described later).
【0018】ヘリコバクターピロリの胃内定着抑制効果 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743を12%脱脂粉乳に0.1
%接種し37℃で24時間培養した。ヘリコバクターピ
ロリが胃上皮細胞であるAGS細胞に定着するとき、こ
の乳酸菌培養液の定着抑制効果を予防及び治療の側面で
測定した。予防の側面でAGS細胞に乳酸菌をまず投与
した後、ヘリコバクターピロリを感染させた場合、ラク
トバシルスアシドフィルスHY 2177とラクトバシ
ルスカセイHY 2743が70%以上の定着抑制率を
示した。治療の側面において、AGS細胞にヘリコバク
ターピロリをまず感染させ、乳酸菌を投与した場合は、
ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY 2743が50%以上の定着抑制
率を示した(後述する実施例3参照)。Gastric colonization inhibitory effect of Helicobacter pylori Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY2743 in 0.1% nonfat dry milk in 0.1%
% Inoculated and cultured at 37 ° C. for 24 hours. When Helicobacter pylori colonized AGS cells, which are gastric epithelial cells, the effect of inhibiting the colonization of the lactic acid bacteria culture was measured in terms of prevention and treatment. When Lactobacillus was first administered to AGS cells from the aspect of prevention and then infected with Helicobacter pylori, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 showed 70% or more inhibition of colonization. In the treatment aspect, if AGS cells are first infected with Helicobacter pylori and lactic acid bacteria are administered,
Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 exhibited a fixing inhibition rate of 50% or more (see Example 3 described later).
【0019】ヘリコバクターピロリ感染により誘導され
るインターロイキン−8生成抑制効果 ヘリコバクターピロリの感染により誘導、生成されるイ
ンターロイキン−8に対し、ラクトバシルスアシドフィ
ルスHY 2177とラクトバシルスカセイHY 27
43の抑制効果をエリザ法(QuantikineTM
HumanIL−8 Immunoassay ki
t, R&D Systems Europe Lt
d.)で測定した。その結果、ラクトバシルスアシドフ
ィルスHY 2177とラクトバシルスカセイHY 2
743がヘリコバクターピロリとともに培養された場合
は、ヘリコバクターピロリ感染により誘導、生成される
IL−8の量をおよそ85%と90%以上抑制すること
が示された(後述する実施例4参照)。Interleukin-8 production inhibitory effect induced by Helicobacter pylori infection Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 27
The inhibitory effect of 43 was determined by the ELISA method (Quantikine ™
HumanIL-8 Immunoassay ki
t, R & D Systems Europe Ltd
d. ). As a result, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2
When 743 was cultured with Helicobacter pylori, it was shown that the amount of IL-8 induced and produced by Helicobacter pylori infection was suppressed by about 85% and 90% or more (see Example 4 described later).
【0020】動物実験によるヘリコバクターピロリ感染
抑制効果確認 ヘリコバクターピロリ感染に対する乳酸菌の予防及び治
療効果を4週齢のBALB/cマウスで確認した。その
結果、ラクトバシルスアシドフィルスHY 2177と
ラクトバシルスカセイHY 2743をまず投与してヘ
リコバクターピロリの感染を予防した場合、ラクトバシ
ルスアシドフィルスHY 2177菌株は生菌数とウレ
アーゼ活性の測定結果、それぞれ93%と90%の予防
率を示した。ラクトバシルスカセイHY 2743の場
合は、生菌数とウレアーゼ活性の測定結果ともに90%
の予防率を示した。Confirmation of Inhibitory Effect of Helicobacter pylori Infection by Animal Experiment The preventive and therapeutic effects of lactic acid bacteria on Helicobacter pylori infection were confirmed in 4-week-old BALB / c mice. As a result, when Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were first administered to prevent infection of Helicobacter pylori, the Lactobacillus acidophilus HY 2177 strain had a viable cell count and urease activity of 93%, respectively. % And 90% protection rates. In the case of Lactobacillus scabii HY 2743, both the viable cell count and the urease activity measurement result were 90%.
Showed a prevention rate.
【0021】また、ヘリコバクターピロリをまず感染さ
せた後、選択された乳酸菌を投与した治療の側面におい
て、ラクトバシルスアシドフィルスHY 2177を投
与した場合、生菌数とウレアーゼ活性の測定結果はそれ
ぞれ87%と83%の治療率を示した。ラクトバシルス
カセイHY 2743を投与して感染されたヘリコバク
ターピロリを除菌した場合は、生菌数とウレアーゼ活性
の測定結果はそれぞれ83%と77%の治療効果を確認
した。In the aspect of treatment in which Helicobacter pylori was first infected and then the selected lactic acid bacteria were administered, when Lactobacillus acidophilus HY2177 was administered, the viable cell count and urease activity were 87% each. And a treatment rate of 83%. When Lactobacillus scabii HY 2743 was administered to remove the infected Helicobacter pylori, the viable cell count and urease activity measured 83% and 77% of the therapeutic effect, respectively.
【0022】したがって、ヘリコバクターピロリの感染
に対してラクトバシルスアシドフィルスHY 2177
とラクトバシルスカセイHY 2743を使用すると、
高い予防と治療効果が得られることを確認した。Therefore, Lactobacillus acidophilus HY 2177 was used against Helicobacter pylori infection.
With Lactobacillus Kasei HY 2743,
It has been confirmed that high preventive and therapeutic effects can be obtained.
【0023】以下、実施例に基づいて本発明をより詳細
に説明する。しかし、つぎの実施例は本発明の範囲を限
定するものではなく、本発明の技術的思想の範囲内で当
業者により通常の変化が可能であるのはもちろんであ
る。Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples do not limit the scope of the present invention, and it is needless to say that ordinary changes can be made by those skilled in the art within the technical idea of the present invention.
【0024】実施例1 ヘリコバクターピロリに対する抗菌活性を有する乳酸菌
の分離 成長剤と発酵乳に使用される乳酸菌を対象としてヘリコ
バクターピロリに対する抗菌活性を比較した。乳酸菌の
ヘリコバクターピロリに対する抗菌活性の測定にはシリ
ンダ法(cylinder method)を使用し
た。乳酸菌は12%スキムミルクに106cfu/ml
の濃度で接種して37℃で24時間培養した。まず、板
当たり四つのシリンダを設け、シリンダの外側にブルセ
ラ寒天を25mlずつ注ぎ凝固させた。109cfu/
mlのヘリコバクターピロリをブルセラブロスに懸濁
し、3mlのブルセラソフト寒天に170μlずつ加え
た。ヘリコバクターピロリを加えたブルセラソフト寒天
を予め製作した培地に注いだ後、37℃のC02培養器
で凝固させた。シリンダを除去し、ホールに乳酸菌培養
液170μlを加えた。37℃のC02培養器で24〜
48時間培養した後、現れた抑制環の大きさを測定、比
較した。この際に、シリンダは直径8.1mmのものを
使用した。Example 1 Isolation of Lactic Acid Bacteria Having Antibacterial Activity against Helicobacter pylori Antibacterial activities against Helicobacter pylori were compared for lactic acid bacteria used for growth agents and fermented milk. The antibacterial activity of lactic acid bacteria against Helicobacter pylori was measured using a cylinder method. Lactic acid bacteria is 106 cfu / ml in 12% skim milk
And cultured at 37 ° C. for 24 hours. First, four cylinders were provided for each plate, and 25 ml of Brucella agar was poured on the outside of the cylinder to solidify. 109 cfu /
ml of Helicobacter pylori was suspended in Brucella broth and added to 3 mL of Brucella soft agar in an amount of 170 μl each. After pouring the prefabricated medium was Brucella soft agar plus Helicobacter pylori, it was coagulated in a C0 2 incubator at 37 ° C.. The cylinder was removed, and 170 μl of a lactic acid bacteria culture was added to the hole. 37. 24 in C0 2 incubator ℃
After culturing for 48 hours, the size of the appeared inhibitory ring was measured and compared. At this time, a cylinder having a diameter of 8.1 mm was used.
【0025】下記の表2に示すように、ラクトバシルス
の場合は、菌株によって違うが、殆どの菌株がヘリコバ
クターピロリに対して抗菌活性を持っていることが確認
された。しかし、ビフィドバクテリウム属とエンテロコ
ッカス属(連鎖球菌属)の場合は抗菌活性が示されなか
った。ラクトバシルスのなかでは、ラクトバシルスアシ
ドフィルスHY 2177が18.9±1.5、ラクト
バシルスアシドフィルスHY 2185が15.6±
1.7、ラクトバシルスカセイHY 2743が14.
2±1.0、ラクトバシルスプランタルムHY 220
7が13.8±0.9の抑制環を示した。したがって、
ヘリコバクターピロリに対して優れた抗菌活性を表す乳
酸菌株としてラクトバシルスアシドフィルスHY 21
77、ラクトバシルスアシドフィルスHY 2185、
ラクトバシルスカセイHY 2743、ラクトバシルス
プランタルム HY 2207を選択した。As shown in Table 2 below, in the case of Lactobacillus, it was confirmed that most strains have antibacterial activity against Helicobacter pylori, although it differs depending on the strain. However, the genus Bifidobacterium and the genus Enterococcus (Streptococcus) did not show any antibacterial activity. Among Lactobacillus, Lactobacillus acidophilus HY 2177 is 18.9 ± 1.5, and Lactobacillus acidophilus HY 2185 is 15.6 ± 1.
1.7, Lactobacillus kassay HY 2743 is 14.
2 ± 1.0, Lactobacillus plantarum HY 220
7 showed an inhibition ring of 13.8 ± 0.9. Therefore,
Lactobacillus acidophilus HY21 is a lactic acid bacteria strain exhibiting excellent antibacterial activity against Helicobacter pylori.
77, Lactobacillus acidophilus HY 2185,
Lactobacillus scabii HY 2743 and Lactobacillus plantarum HY 2207 were selected.
【0026】[0026]
【表2】 [Table 2]
【0027】実施例2 ヘリコバクターピロリウレアーゼ活性抑制効果 抗菌活性により選択した4菌株の乳酸菌を対象としてヘ
リコバクターピロリウレアーゼ活性抑制効果を測定し
た。ヘリコバクターピロリをブルセラ寒天で2日間培養
した後、20mMのPBS緩衝液(pH7.0)で2回
洗浄し、4℃で6分間超音波破砕した。110×gで1
0分間遠心分離した後、上澄み液を収集してヘリコバク
ターピロリ粗−ウレアーゼとして使用した。乳酸菌は1
2%スキムミルクに106cfu/mlの濃度で接種し
て37℃で24時間培養した。乳酸菌のヘリコバクター
ピロリウレアーゼ活性抑制能の比較はまず13.6ml
のウレアブロスに1.6mlの乳酸菌培養液を添加した
後、pHを6.5に補正した。これに800μlのウレ
アーゼを添加し、室温で2時間反応させながら30分間
隔でpHの変化を確認した。Example 2 Helicobacter pylori urease activity inhibitory effect Helicobacter pylori urease activity inhibitory effect was measured on four strains of lactic acid bacteria selected for their antibacterial activity. After Helicobacter pylori was cultured on Brucella agar for 2 days, it was washed twice with 20 mM PBS buffer (pH 7.0) and sonicated at 4 ° C for 6 minutes. 1 at 110 × g
After centrifugation for 0 minutes, the supernatant was collected and used as Helicobacter pylori crude urease. Lactic acid bacteria is 1
2% skim milk was inoculated at a concentration of 106 cfu / ml and cultured at 37 ° C. for 24 hours. The comparison of the ability of lactic acid bacteria to inhibit Helicobacter pylori urease activity was first 13.6 ml.
Was added to 1.6 ml of urea broth, and the pH was corrected to 6.5. 800 μl of urease was added thereto, and the reaction was allowed to react at room temperature for 2 hours, and the change in pH was confirmed at intervals of 30 minutes.
【0028】選択された乳酸菌等に対してヘリコバクタ
ーピロリウレアーゼ活性抑制の効果を測定して図1に示
した。ここで使用したヘリコバクターピロリ粗−ウレア
ーゼの蛋白質の含量は0.52〜0.61mg/ml程
度であった。選択された乳酸菌のヘリコバクターピロリ
ウレアーゼ活性抑制の効果を測定した結果、ラクトバシ
ルスアシドフィルスHY 2177とラクトバシルスカ
セイHY 2743のみがヘリコバクターピロリウレア
ーゼ活性も抑制することが示された。図1に示すよう
に、対照群が反応2時間後にpH8.7程度とウレアー
ゼ活性によるpHが高くなるに対し、ラクトバシルスア
シドフィルスHY 2177とラクトバシルスカセイH
Y 2743がウレアーゼに添加された場合は、反応2
時間後にもpHが6.7程度を維持していた。したがっ
て、ラクトバシルスアシドフィルスHY 2177とラ
クトバシルスカセイHY 2743によりヘリコバクタ
ーピロリのウレアーゼ活性がおよそ90%程度抑制され
ることが確認された。The effect of inhibiting Helicobacter pylori urease activity on the selected lactic acid bacteria and the like was measured and is shown in FIG. The protein content of Helicobacter pylori crude urease used here was about 0.52 to 0.61 mg / ml. As a result of measuring the effect of the selected lactic acid bacteria on the inhibition of Helicobacter pyloriurease activity, it was shown that only Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 also suppress the Helicobacter pylori urease activity. As shown in FIG. 1, the pH of the control group was increased to about 8.7 two hours after the reaction due to the urease activity, whereas Lactobacillus acidophilus HY 2177 and Lactobacillus sarcinate H were increased.
When Y 2743 was added to urease, reaction 2
Even after an hour, the pH was maintained at about 6.7. Therefore, it was confirmed that Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 suppress the urease activity of Helicobacter pylori by about 90%.
【0029】実施例3 AGS細胞を用いたヘリコバクターピロリ胃内定着抑制
の効果 ヘリコバクターピロリに対して抗菌活性効果を示した乳
酸菌4菌株に対してヘリコバクターピロリの胃内定着抑
制の効果をつぎのように予防と治療の観点で確認した。
乳酸菌は12%スキムミルクに106cfu/mlの濃
度で接種して37℃で24時間培養した後、実験に使用
した。まず、予防効果は、AGS細胞(105細胞/ウ
エル)を6培地に付着させ、乳酸菌培養液とRPMI1
640(10%FBS)培地を入れ3時間反応させた。
ヘリコバクターピロリを培地当たり106細胞接種して
1時間付着させ、板シェーカーを用いて3回洗浄した
後、滅菌蒸留水1mlを入れ1時間反応させた。細胞を
収集して遠心分離した細胞ペレットにウレアブロス1m
lを入れ37℃で1時間反応させた後、吸光度(560
nm)を測定した。治療効果は、AGS細胞(105細
胞/ウエル)を6培地に付着させ、ヘリコバクターピロ
リを培地当り106細胞接種し3時間付着させた。乳酸
菌培養液とRPMI1640(10%FBS)培地を入
れ、1時間反応させ、板シェーカーにて3回洗浄した
後、滅菌蒸留水1mlを入れて1時間反応させた。細胞
を収集して遠心分離した細胞ペレットにウレアブロス1
mlを入れ、37℃で1時間反応させ、吸光度(560
nm)を測定した。この際に、対照群としては12%ス
キムミルクを使用した。Example 3 Effect of Inhibition of Helicobacter pylori Gastric Adhesion Using AGS Cells The effect of Helicobacter pylori inhibition of gastric colonization on four strains of lactic acid bacteria showing antibacterial activity against Helicobacter pylori was as follows. Confirmed in terms of prevention and treatment.
The lactic acid bacteria were inoculated into 12% skim milk at a concentration of 106 cfu / ml, cultured at 37 ° C. for 24 hours, and then used in the experiment. First, the preventive effect was that AGS cells (105 cells / well) were adhered to 6 media, and lactic acid bacteria culture solution and RPMI1
640 (10% FBS) medium was added and reacted for 3 hours.
Helicobacter pylori was inoculated with 106 cells per medium and allowed to adhere for 1 hour, washed three times using a plate shaker, and then added with 1 ml of sterilized distilled water and reacted for 1 hour. The cells were collected and centrifuged into a cell pellet with urea broth 1m.
After reacting at 37 ° C. for 1 hour, absorbance (560
nm). Therapeutic effect was as follows: AGS cells (105 cells / well) were adhered to 6 media, Helicobacter pylori was inoculated at 106 cells per media, and allowed to adhere for 3 hours. A lactic acid bacterium culture solution and an RPMI 1640 (10% FBS) medium were added and reacted for 1 hour. After washing three times with a plate shaker, 1 ml of sterilized distilled water was added and reacted for 1 hour. The cells were collected and centrifuged into the cell pellet.
Then, the mixture was reacted at 37 ° C. for 1 hour, and the absorbance (560
nm). At this time, 12% skim milk was used as a control group.
【0030】図2に示すように、予防の側面において、
乳酸菌がまず投与された場合は、ラクトバシルスアシド
フィルスHY 2177とラクトバシルスカセイHY
2743のみが70%以上のヘリコバクターピロリ定着
抑制効果を示し、また、ヘリコバクターピロリが感染さ
れた後乳酸菌が投与された治療的側面においても、ラク
トバシルスアシドフィルスHY 2177とラクトバシ
ルスカセイHY 2743のみが50%以上の定着抑制
効果を示した。したがって、ラクトバシルスアシドフィ
ルスHY 2177とラクトバシルスカセイHY 27
43のみがヘリコバクターピロリに対する抗菌活性、ウ
レアーゼ活性抑制、胃内定着抑制に対する能力を持って
いることを確認した。As shown in FIG. 2, in the aspect of prevention,
When lactic acid bacteria were administered first, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY
Only 2743 shows a Helicobacter pylori colonization inhibitory effect of 70% or more, and also in the therapeutic aspect where lactic acid bacteria were administered after infection with Helicobacter pylori, only Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were used. A fixing suppression effect of 50% or more was exhibited. Thus, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 27
It was confirmed that only 43 had the antibacterial activity against Helicobacter pylori, the ability to inhibit urease activity, and the ability to inhibit gastric colonization.
【0031】実施例4 ヘリコバクターピロリ感染により誘導されるインターロ
イキン−8生成抑制効果 ヘリコバクターピロリの感染により誘導された胃上皮細
胞でのインターロイキン−8の生成に対してラクトバシ
ルスアシドフィルスHY 2177とラクトバシルスカ
セイHY 2743の生成抑制効果を測定した。Example 4 Inhibitory Effect of Interleukin-8 Production Induced by Helicobacter pylori Infection Lactobacillus acidophilus HY 2177 and lactobacillus acidophilus HY2177 inhibited interleukin-8 production in gastric epithelial cells induced by Helicobacter pylori infection. The effect of inhibiting the production of Bacillus scabii HY 2743 was measured.
【0032】ヘリコバクターピロリを10mlのPBS
緩衝液で洗浄した後、ラクトバシルスアシドフィルスH
Y 2177とラクトバシルスカセイHY 2743と
ともに4℃、9,000rpmで10分間遠心分離し
た。それぞれの細胞ペレットを1mlのハンクス液 F
12培地に懸濁し、各懸濁液の少量を取り1:50に希
釈した後、660nmで懸濁度を測定して菌濃度実験条
件に合わせた。Helicobacter pylori in 10 ml of PBS
After washing with buffer, Lactobacillus acidophilus H
The mixture was centrifuged with Y 2177 and Lactobacillus scabii HY 2743 at 9,000 rpm for 10 minutes at 4 ° C. Add 1 ml of Hanks solution F to each cell pellet.
After suspending the suspension in 12 medium, taking a small amount of each suspension and diluting 1:50, the suspension was measured at 660 nm and adjusted to the bacterial concentration experimental conditions.
【0033】細胞集団=16.95×A660×(希釈
因子)×108/ml ヘリコバクターピロリ=1.084×16.95×50
×108=9.18×108 ラクトバシルス=1.408×16.95×50×10
8=9.18×108各培地は下記の4群に分け2回繰
り返して処理した。A群はAGS細胞のみを培養した群
であり、B群はAGS細胞にラクトバシルスアシドフィ
ルスHY 2177とラクトバシルスカセイHY 27
43のみを、C群はAGS細胞にヘリコバクターピロリ
のみを処理した。D群はラクトバシルスアシドフィルス
HY2177とラクトバシルスカセイHY 2743懸
濁液100μlを添加して7.5×108cfu/ml
となるようにし、1時間後にヘリコバクターピロリ懸濁
液100μlを同じ濃度で再び添加した。全ての群に対
し、培養液からそれぞれ0、6、12、24、36、4
8時間にサンプルを取り、エリザ法(Quantiki
neTM Human IL−8 Immunoass
ay kit,R&D System Europe
Ltd.)で測定した。Cell population = 16.95 x A660 x (dilution factor) x 108 / ml Helicobacter pylori = 1.084 x 16.95 x 50
× 108 = 9.18 × 108 Lactobacillus = 1.408 × 16.95 × 50 × 10
8 = 9.18 × 108 Each medium was divided into the following four groups and treated twice. Group A is a group in which only AGS cells have been cultured, and Group B is a group in which AGS cells contain Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY27.
Group 43 was treated with Helicobacter pylori only in AGS cells. Group D was prepared by adding 100 μl of a suspension of Lactobacillus acidophilus HY2177 and Lactobacillus scabii HY 2743, and adding 7.5 × 10 8 cfu / ml.
1 hour later, 100 μl of the Helicobacter pylori suspension was added again at the same concentration. For all groups, 0, 6, 12, 24, 36, 4
Take a sample at 8 hours and use the ELISA method (Quantiki)
neTM Human IL-8 Immunoass
ay kit, R & D System Europe
Ltd. ).
【0034】A:対照群 B:ラクトバシルスアシドフィルスHY 2177(ラ
クトバシルスカセイHY 2743)処理群 C:ヘリコバクターピロリ処理群 D:ラクトバシルスアシドフィルスHY 2177(ラ
クトバシルスカセイHY 2743)+ヘリコバクター
ピロリ処理群 1)サンプリング 板培地(Plate well)内の培地を収集した
後、4℃、12,000rpmで3分間遠心分離した。
上澄み液をエッペンドルフチューブ(eppendor
f tube)に移した後、−70℃で貯蔵した。A: control group B: Lactobacillus acidophilus HY 2177 (Lactobacillus scabii HY 2743) treated group C: Helicobacter pylori-treated group D: Lactobacillus acidophilus HY 2177 (Lactobacillus scabii HY 2743) + Helicobacter pylori Treatment Group 1) Sampling The medium in the plate well was collected, and then centrifuged at 12,000 rpm for 3 minutes at 4 ° C.
Transfer the supernatant to an Eppendorf tube
f tube) and stored at -70 ° C.
【0035】2)標準溶液 RD5P(5×)2.0mlを蒸留水で5倍に希釈した
後、5.0mlを取り、IL−8標準溶液バイアルに添
加して2,000pg/mlのストック(stock)
を製造した。2倍希釈により8濃度の100μl標準溶
液を準備した。2) Standard solution RD5P (5 ×) 2.0 ml was diluted 5-fold with distilled water, 5.0 ml was taken, and added to an IL-8 standard solution vial to prepare a 2,000 pg / ml stock ( stock)
Was manufactured. Eight concentrations of 100 μl standard solution were prepared by 2-fold dilution.
【0036】3)分析(Assay) 分析希釈液(RD1−8)100μlを板培地に入れ、
50μlのサンプルと標準溶液を添加した。 共溶液
(Conjugate)100μlを加えた後、常温で
2時間半の間振動を与えた。板を400μlの洗浄緩衝
液で6回洗浄し、これに試薬Aと試薬Bをそれぞれ1:
1で混ぜ合わせた基質溶液200μlを添加した。板を
30分間定置した後、色変化を観察しつつ492nmで
吸光度を観察した。3) Assay 100 μl of assay diluent (RD1-8) was placed in a plate medium,
50 μl of sample and standard solution were added. After adding 100 μl of the co-solution (Conjugate), the mixture was shaken at room temperature for 2.5 hours. The plate was washed six times with 400 μl of wash buffer, to which Reagent A and Reagent B were respectively added:
200 μl of the substrate solution mixed in 1 was added. After allowing the plate to stand for 30 minutes, the absorbance was observed at 492 nm while observing the color change.
【0037】ヘリコバクターピロリが胃粘膜の上皮細胞
と相互作用するとき、多様なサイトカインの生成を促進
することになり、このなかでもIL−8の生成(sec
retion)が最も著しく表れる。そして、IL−8
は胃炎の直接的な原因となる好中球又はマクロファージ
まで誘導することになる。したがって、ヘリコバクター
ピロリ感染により誘導されるIL−8の生成を抑制する
ことが胃炎発生を抑制するための1方法となり得る。図
3a及び図3bに示すように、ヘリコバクターピロリの
感染有無によって、生成されたIL−8の量に大きな違
いを示した。ヘリコバクターピロリがない培養試料で
は、10時間経過後にもIL−8の生成が殆どないが、
ヘリコバクターピロリ感染菌は2,400〜3,000
pg/ml水準のIL−8の生成が観察された。しか
し、ラクトバシルスアシドフィルスHY 2177又は
ラクトバシルスカセイHY 2743がヘリコバクター
ピロリとともに培養された場合は、ヘリコバクターピロ
リの感染によるIL−8の生成量が400〜700pg
/mlに減少することを確認した。したがって、ラクト
バシルスアシドフィルスHY 2177とラクトバシル
スカセイHY 2743がヘリコバクターピロリの感染
により誘導、生成されるIL−8の量をおよそ85%と
90%以上それぞれ抑制することが理解された。When Helicobacter pylori interacts with the epithelial cells of the gastric mucosa, it promotes the production of various cytokines, and among them, the production of IL-8 (sec)
Ret.) appears most significantly. And IL-8
Will induce neutrophils or macrophages which directly cause gastritis. Therefore, suppressing the production of IL-8 induced by Helicobacter pylori infection may be one method for suppressing the occurrence of gastritis. As shown in FIGS. 3a and 3b, the amount of IL-8 produced was significantly different depending on the presence or absence of Helicobacter pylori infection. In a culture sample without Helicobacter pylori, almost no IL-8 was produced even after 10 hours.
2,400-3,000 Helicobacter pylori-infected bacteria
Production of IL-8 at pg / ml level was observed. However, when Lactobacillus acidophilus HY 2177 or Lactobacillus casei HY 2743 is cultured together with Helicobacter pylori, the amount of IL-8 produced by Helicobacter pylori infection is 400 to 700 pg.
/ Ml. Therefore, it was understood that Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 suppress the amount of IL-8 induced and produced by Helicobacter pylori infection by about 85% and 90% or more, respectively.
【0038】実施例5 動物実験によるヘリコバクターピロリ感染の予防、治療
効果の確認 ヘリコバクターピロリの感染に対する乳酸菌の予防と治
療効果をマウスにより確認した。実験に使用されたBA
LB/cマウスはマウスの胃腸管内に存在してウレアー
ゼ活性を示すヘリコバクターフェリス(Helicob
acter felis)が存在しないものだけを選別
して使用した。使用したBALB/cマウスは4週齢の
雌性で、各群当たり30匹を使用し、25℃、50%湿
度、SPF条件の下に飼育しながら実験した。この際
に、陰性対照群としては、ヘリコバクターピロリ懸濁液
の代わりに7.5%重炭酸ナトリウム緩衝液のみを0.
5mlずつ経口投与した15匹のマウスを使用し、陽性
対照群としては、ヘリコバクターピロリを7.5%重炭
酸ナトリウム緩衝液に2〜4×109cfu/mlの濃
度に懸濁して経口投与した15匹のマウスを使用した。Example 5 Confirmation of Prevention and Treatment Effect of Helicobacter pylori Infection by Animal Experiment The prevention and treatment effects of lactic acid bacteria on Helicobacter pylori infection were confirmed in mice. BA used in the experiment
LB / c mice are located in the gastrointestinal tract of mice and exhibit urease activity.
Only those without acter felis) were selected and used. The BALB / c mice used were 4-week-old females, and 30 mice were used in each group, and the experiment was performed while bred under the conditions of 25 ° C., 50% humidity, and SPF. At this time, as a negative control group, only 7.5% sodium bicarbonate buffer was used instead of the Helicobacter pylori suspension.
Fifteen mice orally administered by 5 ml each were used, and as a positive control group, 15 mice orally administered by suspending Helicobacter pylori in a 7.5% sodium bicarbonate buffer at a concentration of 2 to 4 × 109 cfu / ml. Mice were used.
【0039】1)予防効果 マウスにラクトバシルスアシドフィルスHY 2177
とラクトバシルスカセイHY 2743を飲用水ととも
にそれぞれ一日に2〜5×1010細胞で14日間投与
した後、7.5%重炭酸ナトリウム緩衝液に懸濁した2
〜4×109cfu/mlのヘリコバクターピロリ菌液
を2日間マウス当たり0.5mlずつ6回にわたり感染
させた。感染6週後、各実験群の胃を摘出し、胃組織内
のヘリコバクターピロリ生菌数とウレアーゼ活性の有無
を測定することで、ヘリコバクターピロリ感染に対する
予防効果を確認した。1) Preventive effect Lactobacillus acidophilus HY 2177
And Lactobacillus kassay HY 2743 were administered together with drinking water at 2 to 5 × 10 10 cells / day for 14 days, and then suspended in a 7.5% sodium bicarbonate buffer.
Helicobacter pylori solution of × 4 × 109 cfu / ml was infected for 6 days at 0.5 ml per mouse for 2 days. Six weeks after infection, the stomach of each experimental group was excised, and the preventive effect against Helicobacter pylori infection was confirmed by measuring the number of viable Helicobacter pylori and the presence or absence of urease activity in the stomach tissue.
【0040】2)治療効果 ヘリコバクターピロリを7.5%重炭酸ナトリウム緩衝
液に2〜4×109cfu/mlの濃度に懸濁し、この
菌液を2日間マウスに0.5mlずつ6回にわたり感染
させた。ヘリコバクターピロリがマウスの胃内に完全に
定着した感染6週後、ラクトバシルスアシドフィルスH
Y 2177とラクトバシルスカセイHY 2743を
飲用水とともにそれぞれ一日に2〜5×1010細胞で
3週間投与した。投与後、各実験群の胃を摘出し、胃組
織内のヘリコバクターピロリ生菌数とウレアーゼ活性を
測定することで、ヘリコバクターピロリ感染に対する治
療効果を確認した。2) Therapeutic Effect Helicobacter pylori was suspended in a 7.5% sodium bicarbonate buffer at a concentration of 2 to 4 × 10 9 cfu / ml, and the bacterial solution was infected to mice twice 0.5 ml each for 2 days. Was. Six weeks after infection when Helicobacter pylori was completely established in the stomach of mice, Lactobacillus acidophilus H
Each of Y 2177 and Lactobacillus scabii HY 2743 was administered together with drinking water at 2 to 5 × 10 10 cells / day for 3 weeks. After the administration, the stomach of each experimental group was removed, and the number of viable Helicobacter pylori bacteria and urease activity in the stomach tissue were measured to confirm the therapeutic effect on Helicobacter pylori infection.
【0041】3)胃組織内のヘリコバクターピロリ生菌
数とウレアーゼ活性の測定 摘出した胃組織は、胃内固形分を除去した後、迅速に2
等分して1/2は10%馬血清の添加されたブルセラブ
ロスに浸漬させ、均質化してブルセラ寒天板に分散させ
ておいた。板は湿度の高い10%CO2培養器で37℃
で5〜7日間培養した後、現れたコロニーを確認してヘ
リコバクターピロリの存在の有無を確認する。残りの1
/2はウレアブロスに浸漬させ、湿度の高い10%CO
2培養器で37℃で5日間培養しつつ色の変化を観察し
てヘリコバクターピロリの存在の有無を判断した。3) Measurement of the number of viable Helicobacter pylori and urease activity in the stomach tissue The removed stomach tissue was rapidly removed from the stomach tissue after removing the solid content in the stomach.
An equal portion was immersed in brucella broth supplemented with 10% horse serum, homogenized, and dispersed on a brucella agar plate. The plate is 37 ° C in a humid 10% CO 2 incubator
After culturing for 5 to 7 days, the emerged colonies are confirmed to confirm the presence or absence of Helicobacter pylori. The remaining one
/ 2 is immersed in urea broth and humid 10% CO
The color change was observed while culturing at 37 ° C. for 5 days in two incubators to determine the presence or absence of Helicobacter pylori.
【0042】ヘリコバクターピロリの感染に対するラク
トバシルスアシドフィルスHY 2177とラクトバシ
ルスカセイHY 2743の予防と治療効果を表3に示
した。陰性対照群の場合、感染6週後にもヘリコバクタ
ーピロリが全く感染していないし、一方陽性対照群の場
合は、感染6週後に100%ヘリコバクターピロリ感染
率を示した。しかし、ラクトバシルスアシドフィルスH
Y 2177とラクトバシルスカセイHY 2743を
まず投与してヘリコバクターピロリの感染を予防した場
合、ラクトバシルスアシドフィルスHY 2177菌株
は生菌水とウレアーゼ活性の測定結果、それぞれ93%
と90%の予防率を示した。ラクトバシルスカセイHY
2743の場合は、生菌数とウレアーゼ活性の測定結
果共に90%の予防率を示した。The preventive and therapeutic effects of Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 on Helicobacter pylori infection are shown in Table 3. In the case of the negative control group, Helicobacter pylori was not infected at all even after 6 weeks of infection, whereas in the case of the positive control group, 100% Helicobacter pylori infection rate was shown 6 weeks after infection. However, Lactobacillus acidophilus H
When Y 2177 and Lactobacillus sarcinate HY 2743 were first administered to prevent the infection of Helicobacter pylori, Lactobacillus acidophilus HY 2177 strain showed 93% of live bacterial water and 93% of urease activity, respectively.
And a 90% prevention rate. Lactobacillus Kasei HY
In the case of 2743, both the viable cell count and the urease activity showed a 90% prevention rate.
【0043】また、ヘリコバクターピロリをまず感染さ
せた後、選択された乳酸菌を投与した治療の側面におい
て、ラクトバシルスアシドフィルスHY 2177を投
与した場合、生菌数とウレアーゼ活性の測定結果、それ
ぞれ87%と83%の治療率を示した。ラクトバシルス
カセイHY 2743を投与して、感染されたヘリコバ
クターピロリを除菌した場合は、生菌数とウレアーゼ活
性の測定結果、それぞれ83%と77%の治療効果を確
認した。Further, in the aspect of treatment in which Helicobacter pylori was first infected and then the selected lactic acid bacteria were administered, when Lactobacillus acidophilus HY2177 was administered, the viable cell count and urease activity were 87%, respectively. And a treatment rate of 83%. When Lactobacillus scabii HY 2743 was administered to remove the infected Helicobacter pylori, the viable cell count and urease activity were measured, and as a result, a therapeutic effect of 83% and 77% was confirmed.
【0044】したがって、ヘリコバクターピロリの感染
に対してラクトバシルスアシドフィルスHY 2177
とラクトバシルスカセイHY 2743を使用すると、
高い予防、治療効果が得られることを確認した。Therefore, Lactobacillus acidophilus HY 2177 was tested against Helicobacter pylori infection.
With Lactobacillus Kasei HY 2743,
It was confirmed that high preventive and therapeutic effects were obtained.
【0045】[0045]
【表3】 [Table 3]
【0046】実施例6 菌株混合培養による抗菌活性の相乗効果 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743の混合培養による抗菌活性
の相乗効果をつぎのように確認した。12%スキムミル
クにラクトバシルスアシドフィルスHY 2177とラ
クトバシルスカセイHY 2743を0.1%ずつ接種
し37℃で24時間混合培養した後、培養液によるヘリ
コバクターピロリの抗菌活性を実施例1と同じシリンダ
方法で確認した。そして、各菌株を12%スキムミルク
に0.05%接種し37℃で24時間単独培養液を対照
群として使用し、実験は3回繰り返し実施した。Example 6 Synergistic effect of antibacterial activity by mixed culture of strains The synergistic effect of antibacterial activity by mixed culture of Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY2743 was confirmed as follows. Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were inoculated into 12% skim milk at 0.1% each and mixed and cultured at 37 ° C. for 24 hours. Confirmed by the method. Then, each strain was inoculated 0.05% in 12% skim milk, and a single culture at 37 ° C. for 24 hours was used as a control group, and the experiment was repeated three times.
【0047】図4に示すように、ラクトバシルスアシド
フィルスHY 2177とラクトバシルスカセイHY
2743の単独培養時、ヘリコバクターピロリに対する
抗菌活性はそれぞれ18.7±0.7と15.3±1.
1と表れた。これに対し、ラクトバシルスアシドフィル
スHY 2177とラクトバシルスカセイHY 274
3の混合培養によるヘリコバクターピロリに対する抗菌
活性は19.0±0.8と表れた。その結果、ヘリコバ
クターピロリに対する抗菌活性において、ラクトバシル
スアシドフィルスHY 2177とラクトバシルスカセ
イHY 2743の混合培養が単独培養に比べて相乗効
果をもたらすことが分かった。As shown in FIG. 4, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY
When 2743 was cultured alone, the antibacterial activity against Helicobacter pylori was 18.7 ± 0.7 and 15.3 ± 1.
It appeared as 1. In contrast, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 274
The antibacterial activity against Helicobacter pylori by the mixed culture of No. 3 was 19.0 ± 0.8. As a result, it was found that a mixed culture of Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 has a synergistic effect on the antibacterial activity against Helicobacter pylori as compared with a single culture.
【0048】実施例7 発酵乳製造 脱脂粉乳から無脂乳固形分含量を8〜20重量%に調節
した原料乳を72〜75℃で15秒間殺菌した。殺菌さ
れた原料乳を一定温度まで冷却させた後、ラクトバシル
スアシドフィルスHY 2177とラクトバシルスカセ
イHY 2743を106cfu/mlの濃度で接種し
て、pH4〜5となるまで培養した。培養の完了後、培
養液を冷却させた。一方、果汁濃縮液0.1〜50重量
%、食餌繊維0.1〜20重量%、ブドウ糖0.5〜3
0重量%、オリゴ糖0.1〜15重量%、カルシウム
0.001〜10重量%、ビタミン0.0001〜5重
量%などを溶かしてシロップを製造した。こうして製造
されたシロップを殺菌してから冷却し、前記培養液と一
定の割合で混合、撹拌して均質化させた後、容器に包装
して発酵乳を製造した。こうして製造された発酵乳は、
官能検査の結果、風味、物性、全体的な味において良好
な結果を示した。Example 7 Production of Fermented Milk Raw milk in which the non-fat milk solid content was adjusted to 8 to 20% by weight from skim milk powder was sterilized at 72 to 75 ° C. for 15 seconds. After the sterilized raw milk was cooled to a certain temperature, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were inoculated at a concentration of 106 cfu / ml and cultured until the pH reached 4 to 5. After completion of the culture, the culture was cooled. On the other hand, fruit juice concentrate 0.1-50% by weight, dietary fiber 0.1-20% by weight, glucose 0.5-3%
A syrup was prepared by dissolving 0% by weight, 0.1 to 15% by weight of oligosaccharide, 0.001 to 10% by weight of calcium, 0.0001 to 5% by weight of vitamin and the like. The syrup thus produced was sterilized, cooled, mixed with the culture solution at a predetermined ratio, stirred, homogenized, and then packaged in a container to produce fermented milk. The fermented milk thus produced is
As a result of the sensory test, good results were shown in flavor, physical properties and overall taste.
【0049】実施例8 乳酸菌菌沫の製造 ラクトバシルスアシドフィルスHY 2177とラクト
バシルスカセイHY2743をMRSブロスに106c
fu/mlの濃度で接種し37℃で18〜24時間pH
−調節発酵(pH−control fermenta
tion)を実施した。pH調節は30%NaOHを中
和剤としてpH5.7±0.2で実施した。培養完了
後、4℃で10,000×gで遠心分離して菌体を回収
した。回収された菌体は5%スキムミルクに2.5%乳
漿、5%スクロースが含有された保護剤と同量で混合さ
れた後、凍結乾燥機により粉末化した。こうして製造さ
れたラクトバシルスアシドフィルスHY 2177とラ
クトバシルスカセイHY2743の乾燥菌沫はすべて1
×1011cfu/g以上の生菌数を示した。Example 8 Production of Lactobacillus Bacterial Bacteria Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 were added to MRS broth at 106 c
fu / ml at 37 ° C for 18-24 hours
-Controlled fermentation (pH-control fermenta)
tion) was performed. The pH was adjusted at pH 5.7 ± 0.2 using 30% NaOH as a neutralizing agent. After completion of the culture, the cells were centrifuged at 10,000 × g at 4 ° C. to collect the cells. The recovered cells were mixed with the same amount of a protective agent containing 2.5% whey and 5% sucrose in 5% skim milk, and then powdered with a freeze dryer. The dried bacterial droplets of Lactobacillus acidophilus HY 2177 and Lactobacillus scabytus HY 2743 thus produced were all 1
It showed a viable cell count of × 1011 cfu / g or more.
【0050】実施例9 乳酸菌製剤の製造 実施例7で製造した乳酸菌菌沫から乳酸菌食品、整腸剤
などの乳酸菌製剤を製造した。ラクトバシルスアシドフ
ィルスHY 2177とラクトバシルスカセイHY 2
743の乾燥菌沫20重量%にオリゴ糖10重量%、無
水ブドウ糖20重量%、結晶果糖5重量%、ビタミンC
2重量%、果物粉末香5重量%、アロエ5重量%、食餌
繊維15重量%、車前子(オオバコの種子)の皮18重
量%を混合してスティック又は瓶に一定量分株して包装
した。こうして製造された乳酸菌製剤は5×108cf
u/g以上の生菌数を維持した。Example 9 Production of a lactic acid bacteria preparation A lactic acid bacteria preparation such as a lactic acid bacteria food and an intestinal preparation was produced from the lactic acid bacteria bacterial powder prepared in Example 7. Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2
20% by weight of 743 dried microbes, 10% by weight of oligosaccharides, 20% by weight of anhydrous glucose, 5% by weight of crystalline fructose, Vitamin C
2% by weight, 5% by weight of fruit powder incense, 5% by weight of aloe, 15% by weight of dietary fiber, 18% by weight of skin of psyllium (plantain seeds) are mixed and aliquoted into sticks or bottles for packaging. did. The lactic acid bacteria preparation thus produced is 5 × 108 cf
The viable cell count was not less than u / g.
【0051】[0051]
【発明の効果】以上の実施例から分かるように、本発明
によるヘリコバクターピロリに対して抗菌活性を有する
ラクトバシルスアシドフィルスHY 2177、ラクト
バシルスカセイHY 2743及びそれを用いる乳酸菌
製剤と発酵乳は胃炎、胃潰瘍の原因菌として知られたヘ
リコバクターピロリに対して抗菌活性、ウレアーゼ活性
抑制、胃内定着抑制、インターロイキン−8生成抑制能
力を有する効果があり、ひいてはこれを用いて発酵乳、
乳酸菌食品、整腸薬などの製品に製造して摂取する場
合、ヘリコバクターピロリの感染による胃炎、胃潰瘍、
十二指腸潰瘍の予防と治療が可能な効果を有する。As can be seen from the above examples, Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 having antibacterial activity against Helicobacter pylori according to the present invention, a lactic acid bacterium preparation using the same, and fermented milk are gastritis. Antibacterial activity against Helicobacter pylori, which is known as a causative bacterium of gastric ulcer, has an effect of inhibiting urease activity, inhibiting gastric colonization, and inhibiting the production of interleukin-8.
When manufactured and consumed in products such as lactic acid bacteria foods and intestinal medicine, gastritis, gastric ulcer,
It has the effect of preventing and treating duodenal ulcer.
【0052】また、ラクトバシルスアシドフィルスHY
2177とラクトバシルスカセイHY 2743をと
もに用いた場合は、抗菌活性において有機的な相乗効
果、つまりシナージー効果がある。Also, Lactobacillus acidophilus HY
When both 2177 and Lactobacillus kassay HY 2743 are used, there is an organic synergistic effect in antibacterial activity, that is, a synergistic effect.
【図1】 ラクトバシルスアシドフィルスHY 217
7とラクトバシルスカセイHY 2743によるヘリコ
バクターピロリウレアーゼ活性抑制率の比較結果を示す
グラフ。FIG. 1. Lactobacillus acidophilus HY217
7 is a graph showing the results of comparison of the inhibitory rates of Helicobacter pylori urease activity between Lactobacillus 7 and HY2743.
【図2】 ラクトバシルスアシドフィルスHY 217
7とラクトバシルスカセイHY 2743のヘリコバク
ターピロリ胃内定着抑制を示すグラフ。FIG. 2. Lactobacillus acidophilus HY217
7 is a graph showing the inhibition of Helicobacter pylori gastric colonization by Lactobacillus 7 and HY2743.
【図3】 図3a及び図3bはそれぞれヘリコバクター
ピロリ感染により誘導されるインターロイキン−8生成
に対するラクトバシルスアシドフィルスHY2177と
ラクトバシルスカセイHY 2743の抑制効果を示す
グラフ。FIGS. 3a and 3b are graphs showing the inhibitory effects of Lactobacillus acidophilus HY2177 and Lactobacillus sarcinate HY2743 on interleukin-8 production induced by Helicobacter pylori infection, respectively.
【図4】 ヘリコバクターピロリに対する抗菌活性にお
いて、ラクトバシルスアシドフィルスHY 2177と
ラクトバシルスカセイHY 2743の混合培養による
相乗効果を示すグラフ。FIG. 4 is a graph showing the synergistic effect of a mixed culture of Lactobacillus acidophilus HY 2177 and Lactobacillus scabii HY 2743 on the antibacterial activity against Helicobacter pylori.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) A61P 1/04 A61P 1/04 31/04 31/04 //(C12N 1/20 (C12N 1/20 C12R 1:23) C12R 1:23) (C12N 1/20 (C12N 1/20 C12R 1:245) C12R 1:245) (72)発明者 許▲詰▼成 大韓民国 忠清南道 天安市 新富洞 大 林ハンスプアパート 304−305 (72)発明者 白永振 大韓民国 ソウル市 瑞草区 瑞草3洞 1481−14 賢英ビラ ガ洞 302 (72)発明者 尹錫英 大韓民国 ソウル市 瑞草区 盤浦洞 住 公アパート 3ダンジ 357−405──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) A61P 1/04 A61P 1/04 31/04 31/04 // (C12N 1/20 (C12N 1/20 C12R) 1:23) C12R 1:23) (C12N 1/20 (C12N 1/20 C12R 1: 245) C12R 1: 245) (72) Inventor Xu, Cheng, South Korea Apartment 304-305 (72) Inventor Shiranaga Shinbashi 1481-14 Seocho-dong, Seocho-gu, Seoul, Korea 302 (72) Inventor Youn-Shin-Eng Bukpo-dong, Seocho-gu, Seoul, Korea Residence 3 Danji 357-405
Claims (9)
性、ウレアーゼ活性抑制、胃内定着抑制、IL−8生成
抑制の能力を有するラクトバシルスアシドフィルスHY
2177菌株。1. Lactobacillus acidophilus HY which has an antibacterial activity against Helicobacter pylori, a urease activity, a gastric colonization inhibition, and an IL-8 production inhibition ability.
2177 strain.
性、ウレアーゼ活性抑制、胃内定着抑制、IL−8生成
抑制の能力を有するラクトバシルスカセイHY2743
菌株。2. Lactobacillus scabii HY2743 having an antibacterial activity against Helicobacter pylori, an inhibitory activity of urease, an inhibition of gastric colonization, and an inhibitory activity of IL-8 production.
Strain.
抗菌活性能力を有するラクトバシルスアシドフィルスH
Y 2177菌株とラクトバシルスカセイHY 274
3の菌株が混合された組成物。3. Lactobacillus acidophilus H having a synergistic antibacterial activity against Helicobacter pylori
Y 2177 strain and Lactobacillus scabii HY 274
A composition in which the three strains are mixed.
177菌株を有効成分とすする胃炎、胃潰瘍、十二指腸
潰瘍に効能がある乳酸菌製剤。4. Lactobacillus acidophilus HY 2
A lactic acid bacterium preparation having 177 strains as an active ingredient, which is effective for gastritis, gastric ulcer, and duodenal ulcer.
株を有効成分とする胃炎、胃潰瘍、十二指腸潰瘍に効能
がある乳酸菌製剤。5. A lactic acid bacterium preparation having an effect on gastritis, gastric ulcer, and duodenal ulcer, comprising Lactobacillus scabii HY 2743 as an active ingredient.
177とラクトバシルスカセイHY 2743菌株が混
合されたものを有効成分とする胃炎、胃潰瘍、十二指腸
潰瘍に効能がある乳酸菌製剤。6. Lactobacillus acidophilus HY 2
A lactic acid bacterium preparation which is effective against gastritis, gastric ulcer, and duodenal ulcer, comprising a mixture of 177 and Lactobacillus scabii HY 2743 as an active ingredient.
177菌株を有効成分とする胃炎、胃潰瘍、十二指腸潰
瘍に効能がある発酵乳。7. Lactobacillus acidophilus HY 2
Fermented milk which is effective against gastritis, gastric ulcer, and duodenal ulcer, comprising 177 strain as an active ingredient.
株を有効成分とする胃炎、胃潰瘍、十二指腸潰瘍に効能
がある発酵乳。8. A fermented milk which is effective against gastritis, gastric ulcer and duodenal ulcer, comprising Lactobacillus scabii HY 2743 as an active ingredient.
177とラクトバシルスカセイHY 2743菌株が混
合されたものを有効成分とすする胃炎、胃潰瘍、十二指
腸潰瘍に効能がある発酵乳。9. Lactobacillus acidophilus HY 2
A fermented milk having an effect on gastritis, gastric ulcer, and duodenal ulcer, comprising a mixture of 177 and Lactobacillus scabii HY 2743 as an active ingredient.
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KR2000-7982 | 2000-02-19 |
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